TW201141500A - Use of coconut water extract or coconut shell extract for treating immunological diseases and/or disorders - Google Patents

Abstract

A method for treating immunological diseases and/or disorders associated with tumor necrosis factor-alpha (TNF- α ) are disclosed. The method comprises administering to a subject in need thereof a therapeutically effective amount of the coconut water extract or the coconut shell extract through induction of transforming growth factor beta (TGF- β ) or interleukin-10 (IL-10) as an endogenous immunosuppressive factor.

Description

201141500 VI. INSTRUCTIONS: [Reciprocal References for Related Patent Applications] This application claims priority to US Application No. 61/264,254, filed on Nov. 25, 2009, the entire disclosure of The content is incorporated herein. [Technical Field of the Invention] The present invention relates generally to coconut water or coconut shell extracts and, more particularly, to the treatment of immune diseases and/or abnormalities. [Prior Art] Coconut is a tropical fruit and drink in tropical countries. It has been reported that the antimicrobial and antiviral activity of the shell fiber from coconut is mainly due to the high content of the desired compound (Esquenazi D. et al. 2002). The biological activity of coconut also includes: inhibitory activity against acyclovir-resistant herpes simplex virus type 1 and leishmanicidal effects (Mendonpa-Filho RR. Et al. 2004), hemoglobin to avoid conversion to methemoglobin and free radical scavenging activity due to oxidative reactions caused by nitrite (Alviano DS. et al. 2004). Coconut water is a solution of coconut fruit and has been reported as a refreshing drink that generally and naturally contains fewer side effects. Coconut water is often used as a nutritional formula and food product due to the presence of carb〇hydrates, vitamins, electrolytes, minerals and protein (Santoso U. et ai. 1996 and Campbell-Falck D. et al. 2000). . In addition, 'Coconut water is also used to preserve sheep's follicles and dog's sperm 3 111906 201141500 liquid, goat ovary primary follicles in vitro culture (Figueire (i〇JR 2000 and Andrade ER. et al. 2002), and plants Tissue culture (Tiajnen, τ. 1993). In addition, coconut water has been reported to have inhibitory activity against human bacterial pathogens (Mandal SM. et al. 2009) and has antiulcer effects (Nneli RO. and Woyike OA. 2008). In mammals, the skin is the largest organ of the Integumentary system and acts as a defensive barrier to the invasion of external pathogens. Skin inflammation can evolve into a protective response to injury, thereby eliminating exogenous organisms or substances, and inflammation. The solution mechanism protects us against excessive tissue damage. Failure of the inflammatory mechanism can cause chronic inflammation and lead to many skin diseases. In the murine disease model, it is pointed out that the heterogeneity of TNF receptors is used to suppress autoimmune diseases. Inflamed tissue damage, suggesting that selective TNF receptor inhibition is beneficial for anti-TNF therapy for chronic inflammatory diseases (Apostolaki M, Armaka M, Victoratos P et al. 2010). The amount of tumor necrosis factor a (TNF-a) is associated with increased blister fluid sorption and clearance in psoriatic patients, and is associated with severe disease The degree is proportional (Ettehadi P. et al. 1994, Bonifati C. et al. 1994 and Groves R. et al. 2004). Inflammation can become a protective response to damage or injury, and the original response to inflammation is elimination. Or neutralizing foreign organisms or substances, the inflammatory mechanism of action contains endogenous anti-inflammatory mechanisms that protect us against excessive tissue damage and promote the repair of organizational structure and function. In fact, our health and Survival depends on the efficiency of inflammation and careful balance control. In general, the innate inflammatory response (innate inflammatory 4 111906 201141500 response) can be triggered within a few minutes, if all is sound, the reaction is released within a few hours. Conversely, Chronic inflammation lasts for weeks, even months or years (Lawrence T" Gilroy DW 2007. In the later stages of acute inflammation, white blood cells are produced - 10 and TGF- / 3, to limit inflammation and facilitate resolution mechanisms ^ which two cytokine (IL-10 and TGF-yS) based AND DEVELOPMENT. The mechanism of inflammation is related (Lawrence T. and Gilroy DW 2007 and Serhan CN et al. 2007) ° The liver is at immune endurance due to the anatomical location of the liver, the gastrointestinal tract and the systemic venous circulation. (immunological tolerance) plays an important role. Therefore, it is necessary to avoid an immune response against food or bacterial antigens from the digestive tract. The function of the liver is as a clearing organ, but it hides the risk of tissue damage caused by substances that will be degraded and/or eliminated. Therefore, an effective defense mechanism is necessary. In the non-parenchymal cells Kupffer cells, sinusoidal endothelial cells, and natural killer (NK) lymphocytes, they exert systemic defense functions on the whole body and are suitable for the liver itself. Furthermore, each cell type of the liver, including hepatocytes, has its own defenSe apparatus (Erhardt A. et al. 2010 and Ramadori G. et. al. 2008). The pathological hallmark of rheumatoid arthritis is the inflammation of the synovial fluid in the joint capsule, which forms a pannus, which gradually invades the cartilage and even the hard bone surface. Patients with rheumatoid arthritis have lost their ability to recognize and tolerate self and non-self. The immune system first attacks the synovial tissue and connective tissue of the high-activity joints of the limbs, and then destroys the balance mechanism between bone formation and resorption of 111906 5 201141500, and activates the osteoclasts, thus causing the destruction of articular cartilage and hard bone. . Since the immune mechanism of rheumatoid arthritis involves complex networks linking various cells and cytokines, the detailed pathogenic mechanism of rheumatoid arthritis remains unknown (Gary S. and Firestein, M. 2005). Rheumatoid arthritis or other arthritis-related diseases such as osteoarthritis, ankylosing spondylitis, and acute gouty arthritis include non-steroidal anti-inflammatory drugs (non -steroidal anti-inflammatory drugs, NS AID), corticosteroids, disease modifying anti-Rheumatic Drugs (DMARD), and biological agents. Because arthritis is an inflammatory condition, first-line treatments are aimed at inhibiting inflammation and soothing symptoms, such as NSAIDs and corticosteroids, which are effective in controlling pain, swelling and stiffness associated with arthritis. However, both treatments have side effects and have limited impact on long-term results. Biologics, such as Etanercept, Infliximab, and Adalimumab, are often combined with methotrexate and are also effective in patients with rheumatoid arthritis. However, due to price considerations, it may be considered the second choice. DMARDs, including guanamine pterin, hydroxychloroquine, sulfasalazine, leflunomide, and other antirheumatic drugs such as gold salt and cyclosporine ( Cyclosporine) is a slow-acting compound and its main function is to inhibit the proliferation of immune cells and reduce the inflammatory response of 6 111906 201141500. Although DMARDs are commonly used to diagnose cases of rheumatoid arthritis, the side effects and toxicity reported have been noted (Gaffo A. et al. 2006, Gary S. and Firestein, Μ· 2003 and Gary S. and

Firestein, M. 2005). The complexity and repetitiveness of the combination of regulatory mechanisms with Wecha's interpatient variability, explains why some patients respond to specific treatments, but others do not respond, especially The downstream regulation of immune events is the goal. The cytokine upstream of the drug-specific target for the immune response of rheumatoid arthritis, including IL-1 inhibitors (such as anakinra and kineret), anti-tumor Necrosis factor (anti-TNF-α) agents (eg etanercept, infliximab and adalimumab), co-stimulatory inhibitors of T cell activation (eg abatacept) ), and a selective B cell depletion agent, such as an anti-CD20 monoclonal antibody (eg, rituximab) (Gaffo A. et al. 2006, Gary S, and Firestein, Μ. 2003 and Gary S. and nrestein, M. 2005). These agents can specifically inhibit abnormal immune responses and thus inhibit inflammation, but because of the side effects of immunosuppression, patients are also at high risk of infection. Thus, to date, there has been a need in the art to address the aforementioned deficiencies and inconveniences, particularly in relation to coconut water extracts or coconut shell extracts. SUMMARY OF THE INVENTION In one aspect, the present invention relates to a method for treating an immune disorder or abnormality associated with swollen reliance factor alpha (ΤΝΡ·α) on a subject 111906 7 201141500, including The therapeutically effective amount of the scorpion water extract 1 shell extract 'to induce transformation growth factor 0 (TCFW · 10 (IL-10) as an endogenous immunosuppressive factor. Coconut water extract Wei Yiyi extract by Prepared by the following method: (a) passing coconut water or clam shell water through a resin adsorption type chromatography tube, wherein the coconut shell water is obtained by adding water to the coconut shell; (b) washing with water Tubes; and (c) eluting the column with any solution or combination of solvents capable of desorbing the coconut water extract or coconut shell extract from the column. Coconut water extract or coconut shell extract is effective Treating an immune disease and/or abnormality - the immune disease and/or abnormality is selected from at least one of the group consisting of skin inflammation, liver inflammation, and rheumatoid arthritis. The above and other aspects of the invention will be Combined with the description of the following preferred embodiments The drawings are intended to be illustrative, and may be modified and modified without departing from the spirit and scope of the present invention. The drawings illustrate one or more embodiments of the invention, together with In the written description, the two are used to explain the principles of the present invention. Whenever possible, the reference signs used in the drawings represent the same or similar specific embodiment elements. [Embodiment] The terms used in this specification are The various terms used in the technical scope of the present invention, in the scope of the present invention, and in the specific content, generally have their ordinary meanings. Certain terms used to describe the present invention will be discussed later, or The rest of the specification is discussed as an additional guide for the practitioner to understand the description of the invention. For convenience, certain terms may be emphasized, such as in italics and/or quotation marks. Performance does not affect the scope and meaning of the term, in the same context, regardless of whether the term is emphasized or not, the term The scope and meaning are still the same. It is worth noting that the same thing may be explained in more than one way. Therefore, in this article, alternative language and synonyms are used to represent any one or more terms, regardless of Whether the term is elaboration or discussion in this article, and the use of alternative language and synonyms have no specific meaning. The present invention provides synonyms for certain terms. One or more commonly used synonyms do not exclude others. Use of Synonyms. The examples mentioned in any part of this specification, including examples of any terms discussed, are intended to be illustrative, and do not limit the scope and meaning of the invention or any terms that are described as examples. As such, the invention is not limited to the specific embodiments disclosed herein. The technical or specific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains, unless specifically defined. As for the differences of opinions, this application (including the definitions) will be controlled. As used herein, "about", "about" or "approximate" generally refers to a specified value or range of less than 20%, preferably within 10%, and more preferably within 5%. The numerical quantities mentioned in this paper are only close to the value of 9 111906 201141500, which means that the term "about", "about" or "approximate" can be imagined if not explicitly stated. The exemplary devices, devices, methods, and associated results thereof, which are used in accordance with the present invention, are not intended to limit the scope of the invention. It should be noted that the headings and sub-headings used in the embodiments are only for the convenience of the reader, and do not limit the scope of the invention. In addition, some of the teachings are also described and disclosed below, however, regardless of their ambiguity, the description and disclosure are not intended to limit the scope of the invention. EXAMPLES Example 1 Preparation of Coconut Water Extract or Coconut Shell Extract Coconut (Cocos wwcz/era Linw.) Water and Coconut (10) Tablets (10) Shell Fresh coconut purchased from a fruit vendor (Pingdong County, Taiwan), 247.68 kg (to obtain coconut water), 2.1 kg (to obtain coconut shell). The coconut shell is filled with water, and then broken with a juice machine to obtain coconut shell water. Coconut water (47.6 liters) or coconut shell water (2.7 liters) was filtered through a glass microfiber filter, and the filtrate was continuously passed through a Diaion HP-20 at 4 ° C (Mitsubishi Chemical Co., Japan) ) (52 cm x 5 cm) column chromatography. The residue adsorbed on the gel at 4 ° C was first eluted with deionized water (4 liters, flow rate · 20 ml/min) And then eluted with 100% methanol (ACS grade; Echo Chemical Co Ltd., Taiwan) (about 6.5 liters, flow rate: 8.3 ml/min) until the UV2〇〇 of each eluent- 4()()nm absorption signal detection 10 111906 201141500 Can not be detected. Collect and freeze-dry each deionized water eluate to obtain the water-washed product, which is mainly sugar, weighing 2438.9 g (taken • • coconut water extract) or 13 gram grams (taken from coconut shell extract). Collect 100% methanol extracts, concentrate by vacuum evaporation, and freeze-dry to obtain methanol elution product. In this specification it is called coconut water extract or sputum, shell extract. Chromatography by thin layer chromatography (TCL) to characterize the vinegar content present in deionized water and methanol eluate. The TLC chromatogram is attached to the TLC tablet (Merck, Germany) , Germany)) moving, using 1-butanol: acetic acid: water [3.2:4:0.6 (v/v)] as the eluent, and polyamine TLC tablets (Macherey-Nagel, Germany), using ethyl acetate : pyridine: water [6:3:2 (v/v)] as the eluent. The retention factor (Rf) value of deionized water and ethanol eluate and the sugar standard (D(+) Galactose, D(+) glucose, D(+) mannose, £) (+) arabinose, d(+) ribose, D(+) xylose and maltose; American sigma) for comparison, all sugars It is colored by an anisidine phthalate reagent.

The Rf value and coloration show that the outgoing ion water extract is similar to the glucose and mannose standards, which may indicate that glucose and mannose are the main monosaccharides in coconut water or coconut shell. The results for the 100% sterol eluent showed almost no sugar. Example 2 Effect of coconut water extract or coconut shell extract on lipopolysaccharide-induced RAW 264.7 cell inflammation 11 111906 201141500 RAW 264.7 Cells are cell lines of murine giant scorpion cells and are often used in the study of inflammatory reactions. RAW 264.7 cells (4×1〇4 cells/well) were placed in DMEM high glucose (Gibco) containing 1% (v/v) fetal bovine serum (FBS, Gibco) overnight in a 96-well plate (Falcon). Then in the absence of coconut water extract or coconut shell extract (control group) or in the presence of 0.1, 1 and 10 yg/ml coconut water extract or in the presence of 1, 10 and 100 // g/ml coconut shell water extract 1 # g/ml lip 〇 p〇lysaccharide (LPS; Sigma, USA). The normal group was not treated with LPS nor treated with coconut water extract or coconut shell extract. After a 24-hour incubation period, the nitric oxide (NO) production of the suspension was measured by DAN analysis, and the cell survival rate was measured using a resazurin assay. Nitric oxide (NO) is involved in the inflammatory response and can therefore be used as an indicator to assess the effects of inflammatory reactions. β 2,3-Diaminonaphthalene (DAN, Sigma, USA) and nitrite It can be reduced under acidic conditions to form 1-(H)-naphthotriazole, which is a fluorescent product. A sample of 100 y 1 is first added to the pan. Five DAN freshly prepared DAN (0.01 mg/m b in 〇 62 M HC1) was added and mixed immediately. After incubation at 37 C for 1 minute, the reaction was stopped by the addition of 25 〆丨 28 N of Na〇H. Fluorescence intensity was measured using a fluorescence analyzer (Quoresee Mpiatereader) with excitation light at 355 nm and reading emission at 46 〇 nm (Misko TP. et al. 1993). Results are expressed as mean ± s D (standard deviation) (iv)). The control group represents p< As shown in Figure i, the water extract and the coconut shell extract have similar biological activities, which significantly inhibit the inflammation of RAW 264.7 cells induced by lipopolysaccharide (LPS). 12 111906 201141500 Nitrogen (NO) produce. The ability to differentiate cells can be measured by Rayleigh violet assay (Nociari mm. et al. 1998) 'Resazurin dye is used as an indicator of redox reaction and can be used to detect cell growth but not To measure cell death. Prepare 5 mM Resazurin sodium (Sigma, USA) storage solution in phosphate buffered saline (PBS), and the working solution (5〇gM) is not used from the stock solution. DMEM diluted with DBS high glucose (Gibco). The thunder purple analysis was carried out, the culture solution was removed, and then the freshly diluted thundersha violet working solution was added to each well. Next, the cells were cultured for 2 hours at 37 ° C in an incubator with a water tray (5 〇 / 〇 C 〇 2 _ 95% air), and the Rasha violet dye was reduced to a reduced state by living cells, and the reduction of Rasha The state was detected by a Victor 2 1420 Multilable Counter (Wallac, PerkinElmer) at a fluorescence excitation wavelength of 530 nm and an emission wavelength of 590 nm. The inhibition of LPS-induced inflammatory response by coconut water extract or coconut shell extract (Fig. 1) is not due to a decrease in the number of cells, and coconut water extract or coconut shell extract is not cytotoxic. Example 3 Effect of coconut water extract on TNF-α gene expression in TPA-stimulated HaCaT cells

The HaCaT cell line is a human keratinocyte cell line. HaCaT cells (2.5 X 105 cells/well; in 6-well plates) were cultured in 96-well plates of DMEM high glucose (Gibco) containing 10% (v/v) fetal bovine serum (FBS; Gibco) for 24 hours. The culture was then replaced with DMEM high glucose containing 2% (v/v) fetus 13 111906 201141500 bovine serum (FBS; Gibco) in a 96-well plate and cultured for 24 hours. Then with 2 ng/ml 12-0-tetradecanoyl-phorbol-l3-acetate (TPA, Sigma) in the absence of coconut water extract (control group) Or treatment with 0.1, 1 and 10 β g/ml coconut water extract. The normal group was treated with PBS. After a 6-hour culture period, all of the ribonucleic acid (RNA) was isolated from HaCaT cells using RareRNA reagent (Bio-East, Taiwan) according to the protocol, and reverse transcription was performed using Moloney's murine white blood tumor virus. The enzyme (Moloney murine leukemia virus reverse transcriptase) (Promega) converts RNA into complementary deoxyribonucleic acid (cDNA). Next, quantitative reverse transcription-polymerase chain reaction (RT-PCR) is carried out, and the reaction mixture is generally 20 #b containing lxSYBR GREEN PCR master mix, mixed with 0.5 forward and reverse primers, and 3 ygcDNA. To detect primers: tumor necrosis factor a (TNF-α) (SEQ ID NOs 1 and 2) and GAPDH (sequences 3 and 4) are shown in Table 1. The reaction was performed on a 96-well microtiter plate using the ABI PRISM 7500 Sequence Detector System (Applied Biosystems). The reaction mixture was first incubated at 50 ° C for 2 minutes and at 95 ° C for 10 minutes, followed by 40 cycles of amplification. The step of amplifying the reaction was denaturation at 95 °C for 15 seconds, followed by an indexing at 55 °C for 40 seconds and an expression at 72 °C for 40 seconds. The amount of expression of mouse GAPDH was used as an internal reference. The relative gene expression was calculated by the 2- 14 111906 201141500 △ △ CT method. Results are expressed as mean ± S. D. (n = 2). Compared with PBS. treatment, ##p<0.01. *P < 0.05, **P < 0.01 compared to no TPA. As shown in Figure 2, '0.1, 1, 10 / zg/ml coconut water extract significantly inhibited TPA-induced TNF-α gene expression in TPA-stimulated HaCaT cells. Table 1 Introduction Name Sequence SEQ ID NO: TNF-a Pre-priming 5, -CGG TGC CTA TGT CTC AGC CTC T-3, 1 TNF-a Inverse primer 5, -CAC TCC AGC TGC TCC TCC ACT T-3, 2 GAPDH Pre-priming 5,-GCA AAT TCC ATG GCA CCG T -3' 3 GAPDH Inversion primer 5,-TCG CCC CAC TGA TTT TGG-3, 4 Example 4 Coconut water extract in HaCaT cells for IL-10 and Effect of TGF-Wan Gene Expression HaCaT cells (2.5 X 105 cells/well; in a 6-well plate) were placed in DMEM high glucose (Gibco) containing 10% (v/v) fetal bovine serum (FBS; Gibco). 96-well plate (Falcon) for 24 hours. The culture was then replaced with DMEM high glucose (Gibco) containing 2% (v/v) fetal calf serum (FBS; Gibco) in a 96-well plate (Falcon) and cultured for 24 hours. It was then treated with 0·1, 1 15 111906 201141500 and 10 yg/ml coconut water extract, and the normal group was treated with pBS. After 6 hours of culture, use RareRNA reagent (Bi〇_East, Taiwan) to separate all ribonucleic acid (RNA) from HaCaT cells according to the protocol, and use Moloney's murine white blood tumor virus reverse transcriptase (Promega) Transforming RNA into complementary deoxyribonucleic acid (cDNA). Next, quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with a total mixture of 20 " containing lxSYBR GREEN PCR master mix, mixed with 0.5 forward and reverse primers, and 3 egCDNA. To detect primer pairs: interleukin 10 (IL-10) (SEQ ID NOs: 5 and 6), transforming growth factor cold (TGF-dots) (SEQ ID NO: 7 and 8), and GAPDH (SEQ ID NO: 9 and 10) are shown. In Table 2. Reactions were performed in a 96-well microtiter plate using an ABI PMSM 7500 Sequence Detection System (Applied Biosystems). The reaction mixture was first incubated at 50 ° C for 2 minutes and at 95 ° C for 1 Torr, followed by 40 cycles of amplification. The step of amplifying the reaction was denaturation at 95 ° C for 15 seconds, followed by adhesion at 55 ° C for 40 seconds, and at 72 ° C for 40 seconds. The amount of expression of mouse GAPDH was used as an internal reference. The relative gene expression was calculated by the 2·ΔΔ CT method, and the result was expressed as the mean S.D. (n=2). Compared with PBS alone, < 〇.〇卜 As shown in Figure 3, coconut water extract has a tendency to induce IL-10 (Fig. 3A) and TGF-call (Fig. 3B) gene expression in HaCaT cells. . 16 111906 201141500 Table 2 Introduction Name Sequence Serial Number IL-10 Pre-introduction 5, -GCC TAA CAT GCT TCG AGA TC-3, 5 IL-10 Inverse primer 5'-TGATGT CTG GGT CTT GGT TC-3, 6 TGF -/3 pre-priming 5,-AAA TGG ATA CAC GAA CCC AA-3, 7 ΎβΈ-β inverted primer 5'-GCT GCA TTT GCA AGA CTT TAC-3, 8 GAPDH pre-priming 5,-GCA AAT TCC ATG GCA CCG T -3, 9 GAPDH Inverse primer 5'-TCG CCC CAC TGATTT TGG-3, 10 Example 5 Effect of coconut water extract on TPA-induced ear skin inflammation model 8-week-old female BALB/c small gas system Purchased from the National Experimental Research Institute: Animal Testing Center (NLAC, Taiwan). In order to induce γ mouse strain in the mouse ear, the following formula was taken: in the absence of coconut material, or in two days. .2 small 5_'s Nvzi water (2: Apply 8〇_12_〇_14: linol-13-acetic acid vinegar (TPA, Sigma) to the skin of the mouse. The normal group is treated with pBs. Ugly 111906 17 201141500 After 6 hours and 36 λ of TPA treatment, the mouse ear skin thickness was recorded (Fig. 4). The results are expressed as mean ± SD (n = 2 to 6). Compared with the normal group ' # >< 0.05, _P < 〇〇〇b compared with τρΑ only, < 0.05, **P < 〇.〇1, < 〇〇〇卜 as shown in Figure 4, at 6 After 5 hours of TPA treatment, '5 mg/kg of the water extract of the scorpion had a significant inhibitory effect on the inflammatory response of the mouse ear-induced skin, and after 36 hours of TPA treatment, by 〇2, 】, 5 The water extract also significantly inhibited the inflammatory response of TPA-induced mouse ear skin. After 36 hours of TPA treatment, the bio-slice of mouse ear skin was stained with hematoxylin and eosin (Hemat〇xylin) And E〇sin) (h&e)

Staining analysis (Fig. 5A to 5J). The magnifications of the 5th to 5e graphs are 200 times, and the magnifications of the 5F to 5J maps are 4 times. As shown in Figures 5B and 5G, 80 y Μ TPA treatment significantly induced skin edema in mouse ears compared to the normal group (Figures 5A to 5F). In addition, 〇2mg/kg (5C and 5H), 1 mg/kg (5D and 51), and 5 mg/kg (5E and 5J) coconut water extract treatment significantly inhibited τρΑ-induced skin edema And reduce the infiltration of inflammatory cells. Example 6 Effect of coconut water extract on lipopolysaccharide-induced hepatocyte inflammation C57BL/6J Narl mice, 8 to 10 weeks old, were purchased from the National Experimental Research Laboratory Animal Center (NLAC, Taiwan). Modified by methods described in the previous literature to isolate hepatocytes (Kreanier BL. et al. 1986, 18 111906 201141500

Kojima T. et al. 2001 and Liu S. et al. 2002). The mice were anesthetized with avertin (into 6 out of 11) (2% (review / ¥) 2,2,2-tribromoethanol, 4.30 〇 1118 / 1 ^ body weight), followed by containing 0.5 mM ethylene glycol - Bis (2-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA; Sigma-Aldrich) Hank's balanced salt solution (HBSS) in the inferior vena cava Liver perfusion technology. The perfusate flowed out through the severed hepatic portal vein and its flow rate was 1 ml/min. After 10 minutes of perfusion, 0.05% (w/v) collagenase (Type II; Worthington Biochemical Corporation) and 1 mM Sigma-Aldrich HBSS solution were added to the perfusion device. Perfuse at the same flow rate for ten minutes. A portion of the digested liver was removed and placed in a 60 mm plate (BD Falcon) containing HBSS and slightly shredded to allow hepatocytes of the hepatic sinus to be released. The cut liver suspension was filtered through a nylon membrane (300 pore size), followed by centrifugation at 50 x g for 3 minutes to obtain a cell pellet. The cell pellet was suspended in DMEM medium (Gibco/Invitrogen) and centrifuged at 50 x g for 5 minutes in 35% (v/v) percoll (GE Healthcare) medium. The cell pellet was further washed twice or three times with DMEM medium, and then centrifuged at 50 x g for 3 minutes to obtain hepatocytes. The isolated hepatocytes were placed in a 96-well plate (Costar) containing 10% FBS (Gibco) in DMEM medium at approximately lxlO4 cells/well, and then in an incubator (5% C02-95% air) with a water tray. Incubate at 37 °C. Hepatocytes were then contained at 1 or 10 //g/ml lipopolysaccharide (LPS; Sigma, USA) in the absence of coconut water extract (control group) or in the presence of 0.1 and 1 yg/ml coconut 19 111906 201141500 water extract. 2〇/0 (v/v) FBS (Gibc〇) in DMEM was cultured to septum. The normal group was not treated with LPS nor with coconut water extract. After a 24-hour incubation period, the nitric oxide (NO) production of the suspension was measured by DAN analysis and the cell viability was measured using a Rasal assay. NO participates in the inflammatory response of the liver and can therefore be used as an indicator to assess the effects of an inflammatory response. 2,3-Diaminonaphthalene (DAN, Sigma, USA) and nitrite can be reduced under acidic conditions to form an old naphthacene triazole, which is a fluorescent product. Add 1 〇〇 from the 丨 sample to the plate first. Add 5 〇 freshly prepared DAN (〇.〇5 mg/mi in 〇 62 μ HC1) and mix immediately. After incubation for 1 min at 37 C, the reaction was stopped with 25"12 8 〇 Na〇H. The excitation light was excited with 355 ηιη using a fluorescence analyzer and the emission light at 460 nm was read to measure the fluorescence intensity (Misk〇) Τρ. et al. 1993). The results are expressed as mean ± s D (n = 6 b compared to the control group, *** means p < 0.001. As shown in Figure 6, in 〇 1 and 1 # g /mi's coconut water extract significantly inhibits nitric oxide production induced by L ps inflamed cells. The ability of cell differentiation can be measured by Rayleigh violet analysis (N〇dari MM et al. 1998) Sand violet dye is an indicator of redox reaction, which can be used to detect cell growth, but can not be used to measure cell death. Prepare 5 mM Rasha i#3 (Sigma, USA) storage solution in saline buffer physiological saline (PBS), and the working solution (5 〇# M) was diluted from the stock solution using DMEM without FBS. The Rasha violet analysis was performed, the culture solution was removed, and then the freshly diluted Rasha violet working solution was added. In each well, then in an incubator with a water tray (5% C〇2_95% air) at 37»c 1906 20 201141500 For 2 hours, the Rasha violet dye was reduced by the activity of living cells, and the reduced state of Rasha violet was stimulated by a Victor 2 1420 Multilable Counter (Wallac, PerkinElmer) at 530 nm and emission wavelength. Detection at 590 nm. The liver cell line administered with coconut water extract is not cytotoxic. Therefore, coconut water extract is effective in treating inflammatory reactions. Example 7 Coconut water extract induced in D-GalN/LPS Effect of liver inflammation on liver injury Male C57BL/6J Narl mice, 8 weeks old, were purchased from the National Experimental Research Laboratory Animal Center (NLAC, Taiwan). Preparation of LPS/D-GalN induced acute liver inflammation (Yamada I. et al 2008), mice were injected intraperitoneally (ip) with D-aminogalactose (D-GalN; Sigma, USA) (600 mg/kg body weight) and lipopolysaccharide (LPS; Sigma, USA) (8 pg/kg body weight) Phosphate buffered saline solution. Oral administration of coconut water extract (0.2 and 1 mg/kg body weight) dissolved in boiling water 24 hours and 30 minutes before LPS/D-GalN injection, the control group gave Is equal to boiled water. Normal group is injected intraperitoneally D-GalN LPS injection and the same amount of PBS. The combined after the injection of LPS / D-GalN 3 hours, the serum samples were collected and liver tissues, respectively Serum transglutaminase activity and the amount of mRNA. By measuring Alanine Aminotransferase (ALT) (also known as Glutamic pyruvic transaminase (GPT)) and Aspartate aminotransferase (AST) (also known as Aspartate aminotransferase (AST)) Glutamic oxaloacetic transaminase (GOT), Jinqing Enzyme Activity 21 111906 201141500 Sex 'GPT (ALAT) IFCC mod. and GOT (ASAT) IFCC mod· (HUMAN GmbH, Germany) The group was evaluated according to the method of operation to assess liver damage. Results are expressed as mean to 6). Compared with the control group, ** means p < 〇.〇1 and *** means p < 〇〇 (Π. As shown in Figures 7 and 7Β, after D-GalN/LPS induces liver inflammation, 〇 The .2 mg/kg coconut water extract significantly inhibited the amount of GPT, while the 1 mg/kg coconut water extract also significantly inhibited GOT and GPT levels. Using RareRNA reagent (Bio-East, Taiwan) according to the instructions for use. The liver tissue is isolated from all ribonucleic acid (RNA) and the RNA is converted to complementary deoxyribonucleic acid (cDNA) using Moloney's murine white blood tumor virus reverse transcriptase (Promega). The quantitative reverse transcription polymerase chain reaction is then performed ( RT-PCR), the total volume of the reaction mixture was 20 to 1, including lxSYBR GREEN PCR master mix, mixed forward and reverse primers of 0.5 /z Μ, and 3 /zg cDNA. To detect primer pair: tumor necrosis Factor alpha (TNF-alpha) (SEQ ID NO: 11 and 12) and /3 actin (SEQ ID NO: π and 14) are shown in Table 3. This reaction was performed on a 96-well microtiter plate with the ABI PRISM 7500 Sequence Detector System ( Applied Biosystems) The reaction mixture was first incubated at 50 °C for 2 minutes and at 95 °C for 1 minute, then proceeded 40 cycles of amplification reaction. The steps of this amplification reaction were denaturation at 95 ° C for 15 seconds, followed by adhesion at 55 ° for 40 seconds and at 72 ° for 30 seconds. The expression of mouse cold actin was used as an internal reference. The expression amount of the gene was calculated by the 2-ΔΔσΓ method. The result was expressed as the mean soil SD (n=4 to 6). Compared with the control group, '*** indicates p < 0.001. As shown in Fig. 7C, 0.2 And 1 mg/kg of coconut water extract significantly inhibited the TNF-α expression of liver inflammation induced by 22 111906 201141500 D-GalN/LPS. According to the results of Figures 7A to 7 C, coconut water extract was administered. Inflammation of the liver (impaired) has therapeutic effects. In addition, the TNF-α inhibitory effect of coconut water extract can be used to treat immune diseases and/or abnormalities associated with TNF-α. Table 3 Introduction Name Sequence Serial Number TNF- a before Introduction 5, -CCA GGC AGT CAG ATC ATC TTC TC-3, 11 TNF-a Inverse primer 5 '-AGC TGG TTA TCT CTC AGC TCC AC -3 ' 12 square actin preamplifier 5,-GTG GGC CGC CCT AGG CAC CA-3' 13 Calling Actin Inversion Primer 5'-TGG CCT TAG GGT TCA GGG GG-3, 14 Example 8 Effect of Coconut Water Extract on Survival Rate of D_GalN/Lps-Induced Liver Inflammation in Mice Male-female C57BL/6J Narl mice at 9 weeks of age were purchased from the National Experimental Research Laboratory Animal Center ( NLAC, Taiwan). Preparing LPS/D-GalN to induce acute hepatic inflammation (Yamadal. et al. 2008), mice were injected intraperitoneally (ip) with D-aminogalactose (D-GalN; Sigma, USA) (600 mg/kg body weight) and month曰 酿 (LPS; Sigma, USA) (8 pg / kg body weight) of the salt buffer 23 111906 201141500 physiological saline. At 24 hours and 30 minutes before the combined injection of LPS/D-GalN, the coconut water extract (〇2, 1 and 5 mg/kg volume) dissolved in boiling water was orally administered twice, and the control group was given The amount of boiling water. The normal group was also intraperitoneally injected with the same amount of PBS as D-GalN and LPS injections. As shown in Figure 8, 0.2, 1 and 5 mg/kg coconut water extract significantly reduced mortality in D-GalN/LPS-induced liver-inflamed mice. Example 9 Preparation of Proteoglycan Proteoglycan Proteoglycans were prepared by reference to previous literature conditions (Finnegan A. et al. 1999 and Giant TT. and Mikecz K. 2004.). Briefly, cartilage sections were frozen at _70 °C 'milled using a pulverizer, then 4 guanidine hydrochloride (Sigma, USA), 10 mM EDTA (Sigma, USA), 2 mM benzene Phenyl 醯 醯 ( I (phenylmethylsulfonyl fluoride; PMSF; Sigma, USA), 2 mM iodoacetamide 0 〇廿〇 3 〇 6 {311 ^ 廿 6; 81 layers 11 ^, 1; 8 into) and 5 (( ^/1111 pepsin inhibitor saponin 313 was extracted at 11 8; 8 丨 8 〇 1 amp 1 & 1, 8 VIII. 4. After 24 hours, centrifuged at 2000 x g for 40 minutes, and the extract was collected to obtain Suspension. The suspension was further centrifuged at 75000 xg for 48 hours at 4 °C in a gradient of chlorinated (1.5 g/ml; Sigma, USA) to collect fractions with a density greater than 1.56 g/ml. Dialysis at 4 °C After dialysis with 0.1 Μ sodium acetate (Sigma, USA) at pH 7.4 (using a dialysis membrane (snake skin pleated dialysis tube 3500 MW, Pierce) and deionized water for four times, the fraction was freeze-dried to obtain proteoglycan Crude extract, yield 0.51%. 24 111906 201141500 The crude proteoglycan extract was dissolved in 50 mM Tris at pH 8.0 (UBS, U SA) and 60 mM sodium acetate (Sigma, USA) buffer, then hydrolyzed with chondroitinase (chodroitinase ABC; luint; Sigma, USA) for 24 hours at 37 ° C. After hydrolysis, the buffer was adjusted to pH 5.8, Further adding keratinase (keratinase; 5.6 uints; Sigma, USA), hydrolysis at 37 ° C for 24 hours, and finally the crude extract of proteoglycan, deionized Ηβ dialysis and freeze-drying to obtain de-glycosylated proteoglycan (consumption) The proteoglycans of the glycosaminoglycan side chain are named proteoglycan in this specification. The proteoglycans are deuterated by 12 〇/〇 denatured dodecyl sulfate nanopolyacrylamide gel electrophoresis (Sodium dodecyl) Sulfate-polyacrylamide gel electrophoresis; SDS-PAGE, data not shown) Analysis and determination. The yield of proteoglycan was about 19.13 〇 / 0. Example 10 Effect of coconut water extract on proteoglycan-induced arthritis model The induced arthritis model (rheumatoid arthritis model) was modified with reference to the literature conditions (Finnegan a. et al. 1999 and Giant TT. and Mikecz Κ 2004). Female BALB/c mice from 8 to 10 weeks of age were purchased from the National Experimental Research Laboratory Animal Center (NLAC, Taiwan). For the proteoglycan-induced joint inflammation group, the mice were immunized by intraperitoneal injection of proteoglycan emulsion (phosphate containing 1 or 5 g of proteoglycan) on sputum, 14, 28, 42 and 63 days. Buffered physiological saline, and 1 mg of dimethyldioctadecylammonium bromide» DDA; Sigma, USA 25 111906 201141500 adjuvant). From day 14 to the end of the experiment, 0.2, 1 and 5 mg/kg/day of coconut water extract (0.2, 1 and 5 mg/kg/day, respectively) was added to the feed. In the normal group, PBS was injected intraperitoneally and the mouse standard diet (LabDiet®) was administered. The mice were sacrificed on day 77. At the end of the experiment, the incidence of the 5 mg/kg/day group and the thickness of the sole of the foot were rapidly increased as the control group, and the final incidence was about 85% (Figures 9A and 9B). The final incidence of the 1 mg/kg/day group was approximately 40% (Figures 9A and 9B). In the 1 mg/kg/day group, the thickness of the sole of the foot was only slightly swollen, and after the last proteoglycan injection, the paw swelling was slowed down on the 73rd day (Fig. 9A). The swelling of the soles of the 1 mg/kg/day group and the clinical rheumatoid arthritis were slower than those of the three groups of coconut water extracts (Figures 9A and 9B). After the last injection of proteoglycan at the end of the experiment, the incidence of the 0.2 mg/kg/day group increased to approximately 50%. After the last injection of proteoglycan, the swelling of the paw of the 〇·2 mg/kg/day group increased, but began to slow down at 73 days' and was similar to the 1 mg/kg/day group (Fig. 9A). In addition, the clinical signs of foot thickness and rheumatoid arthritis were significantly lower during the trial than in the control group (Figures 0A and 9B). Coconut water is used in nutritional formulas and food products. It is commonly used in folk medicine to slow down fever and treat gastroenteritis, bladder stones and coronary heart disease (Mandal SM. et al. 2009). In addition, coconut water has been reported to inhibit human bacterial pathogen activity and anti-ulcer effects (Mandal SM. et al. 2009 and Nneli RO. and Woyike OA. 2008). Therefore, there are still many unknown effects on the immune response. The present invention has proved that coconut water extract can inhibit the production of TNF-α and NO 26 111906 201141500, which is related to inflammation or autoimmune diseases and can induce the production of IL-10 and TGF- /5. Reducing the inflammatory response (Lawrence T. and Gilroy DW2007 and Serhan CN et al. 2007). In addition, 'coa a water extract has therapeutic effects on skin inflammation, liver inflammation and rheumatoid arthritis, especially at low doses (eg below 2 mg/kg/day), so it is immune to diseases and/or abnormalities. It has immunosuppressive effects such as inflammation and autoimmune diseases. The present invention demonstrates that coconut water or coconut shell extract can be used to treat immune diseases and/or abnormalities and to inhibit the clinical symptoms of inflammation. For arthritic mice, the therapeutic dose of 2 mg/kg/day is equivalent to 0.162 mg/kg/day for humans at 60 kg body weight (Reagan-Shaw S. et al. 2008). A hazelnut fruit (containing about 500 ml of coconut water) gives 20 mg of coconut water extract. For humans, the therapeutic dose of 2 mg/kg/day coconut extract is about the same as that of 243 ml of coconut per day. Water, about half of coconut fruit's is similar to the amount of folk medicine used. Therefore, in a general immunosuppressive treatment, even a high dose (e.g., a 50 mg/kg/day coconut water extract) does not cause side effects such as suppression of immune cell proliferation and hematopoietic function. Many immune diseases and/or abnormalities still need to be treated by anti-inflammatory or immunosuppressive treatments, such as skin inflammation, liver inflammation, arthritis (such as rheumatoid arthritis, osteoarthritis, multiple nodular arteritis and Acute gouty arthritis), allergies (such as asthma, eczema (atopic dermatitis), allergic rhinitis and eye allergies), condensed globulinemia, immune platelet deficiency, purple spot, systemic vasculitis, autologous Immune hemolytic poor 27 111906 201141500 Blood, Raynaud's syndrome, systemic lupus erythematosus, scleroderma (systemic sclerosis), non-insulin-dependent diabetes mellitus (IDDM), inflammatory bowel disease (eg cloning) Disease (Koo AP. 2000, Davidson A. and Diamond B. 2001 and Kim ΕΥ. and Moudgil KD. 2008), septicemia and septic shock (Annane D. et al. 2009), cognac (Menter A) Et al. 2009) and Bethel's disease (Sakane T. et al. 1999). Therefore, administration of coconut water extract or coconut shell extract is beneficial to the above-mentioned immune diseases and/or abnormalities without the disadvantages of anti-inflammatory or immunosuppressive treatment. In addition, the present invention demonstrates that coconut water extract or coconut shell extract inhibits TNF-α. TNF-α is a cytokines that produce a variety of cellular metamorphosis, including monocytes and macrophages, which were originally thought to cause tumor necrosis in some mice. A series of analyses of the effects of TNF-[alpha] demonstrate that TNF plays an important role in the network of pro-inflammatory cytokines. Many chronic inflammations are characterized by white blood cell aggregation, activation and proliferation, and mediators of inflammation that are associated with TNF (Tracey D. et al. 2008). TNF-α is involved in many pathogenic mechanisms of inflammatory diseases and abnormalities, and is treated with anti-TNF-α, such as rheumatoid arthritis, dryness and dryness arthritis, juvenile chronic arthritis, ankylosing spondylitis, inflammatory bowel Diseases (including Crohn's disease), ulcerative colitis, Bessel's disease, juvenile idiopathic arthritis, uveitis (Valesini G. et al. 2007, Wong M. et al. 2008 and Lin J· Et al. 2008), adult schizophrenia, Wegener granulomatosis, scleroderma, sedative syndrome, sarcoma, gangrenous pyoderma and polymyositis and dermatomyositis (Tutuncu Z. Et al. 2002). Therefore, coconut water extract or clam shell extract can be used to treat these TNF-α related diseases and/or abnormalities. The above description of a field can be seen as a description of the actual &amplifications, but without departing from the precise modifications of the invention. Therefore, it is not intended to limit the invention except as to the accompanying application. The foregoing examples are illustrative of the principles of the present invention and the actual application of the present invention so that the present invention can be applied to the specific use of other techniques. The manner in which the implementation is changed is foreseeable without departing from the spirit and scope of the invention: therefore, the scope of the invention as defined in the present invention is not the above description and the examples. References cited and discussed herein include patents, patent applications, and published literature. The materials referred to and/or discussed in the present invention are intended to be illustrative only and not to be considered in the scope of the prior art. All references and discussion references in this specification are hereby incorporated by reference in their entirety in their entireties in the the the the the the the The references are listed below:

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Kreamer BL. et al. 1986. In Vitro Cell Dev Biol. 30 111906 201141500 22:201-211. . Lawrence T. and Gilroy D.W.2007. Int J Exp Pathol. 88:85-94

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Reagan-Shaw S. et al. 2008. FASEB J. 22:659-661. S. Kintzios. et al. 2000. Scientia Hort. 85:137-144. Santoso U. et al. 1996. Food Chem. 57: 299-304. SakaneT. et al. 1999. N Engl J Med. 341:1284-1291. Serhan C.N. et al. 2007.^5^7 21:325-332 Tiainen, T. 1993. Plant Sci. 88 :83-90. 31 111906 201141500

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Wong M. et al. 2008. Clin Immunol. 126:121-136. Yamada I. et al. 2008. Cytokine. 41:293-301. [Simplified Schematic] Figure 1 shows a coconut water extract Or the effect of coconut shell extract on the inflammation of RAW 264.7 cells induced by LPS. The control group was administered lipopolysaccharide (LPS) to induce inflamed RAW 264.7 cells. The treatment group was administered LPS containing 0.1, 1 and 10 g/ml coconut water extract or 1, 10 and 100 / z g/ml coconut shell extract. The normal group was not treated with LPS nor treated with coconut water extract or coconut shell extract. The production of nitric oxide (NO), an indicator of inflammation, was measured by DAN analysis. The results are expressed as mean ± 8.0. (n = 6). * indicates p < 0.05. Figure 2 is a graphical representation showing the effect of coconut water extract on THA-stimulated HaCaT cells for TNF-α gene expression. Keratinocyte cell line HaCaT cells (2.5 x 105 cells/well; in 6-well plates) containing TPA (2 ng/ml) with different concentrations of coconut water extract (0, 0.1, 1 and 10 V g/ml) Culture in culture medium. The mRNA concentration of TNF-α was analyzed by real-time PCR after a 6-hour culture period of TPA and coconut water extract. The results are expressed as mean ± S. D. (n = 2). Compared with the PBS treatment group ##p<001. *P<0.05, **P<0.01 compared to the TPA-free group. Figure 3 is a graphical representation showing mRNA expression in HaCaT cells induced by coconut water extracts 111906 32 201141500 IL-10 (Fig. 3A) and TGF-dots (Fig. 3B). The keratinocyte cell line HaCaT cells (2.5χ105 cells/well; in a 6-well plate) were cultured in a culture medium containing scorpion water extracts (〇, 〇·1, 1 and 1 〇β g/m〇). After 6 hours of culture period of coconut water extract, the mRNA levels of IL-10 and TGF-/? were analyzed by real-time PCR. The results were expressed as mean ± SD· (n=2). Only compared with PBS group** ρ<〇.〇1. The diagram in Figure 4 shows the effect of coconut water extract on TPA-induced mouse ear thickness. TPA 80# 上 The appearance of the upper skin of the mouse ear results in the induction of skin water cavity' and Two days of pretreatment with Nippon water extract (〇·2, 1 and 5 mg/kg/day) inhibited the induction of skin edema in a dose-dependent manner. The results were expressed as mean ± 8.0. Compared with the normal group #p <0.05,#"Ρ<0·001. Only as compared with the TPA-treated group *P<0.〇5, **P<0.01, ***P<0.001° Figure 5 The effect of coconut water extract on TPA-induced ear edema and cell infiltration is shown. The magnification of Figure 5A to Figure is 200 times, and that of Figure 5F to 5J is 400 times. The ear skin biopsy is local. TPA (80/zM) was collected after 36 hours of treatment. Different doses of coconut water extract (G.2, 1 and 5 mg/kg/day) were orally pretreated for two days to inhibit the induction of skin edema. The inflammatory effect of inflammatory cells is reduced after TPA treatment. The illustration in Fig. 6 shows the effect of coconut water extract on the inflammation of hepatocytes induced by lipopoly vinegar. The control group is a liver cell that induces inflammation and induces inflammation. LPS is given in the presence of 〇1 and] / * and 1 Μ/ΙΏ1 coconut water extract. The normal group is not treated with lps nor with coconut water extract at 111906 33 201141500. DAN analysis Nitric oxide (NO) production, inflammation index. The results are expressed as mean soil SD (n=6). *** indicates p<〇〇〇1 0 Figure 7A shows coconut water extract in D- GalN/LPS induces the effect of liver inflammatory mouse model on liver injury. The control group is administered D-GalN and lipopolysaccharide (LPS) to induce liver inflammation. The normal group is given phosphate buffered physiological saline. Water (PBS) instead of D-GalN and LPS, and fed with coconut water extract at doses of 0.2 and 1 mg/kg/day Determine the activity of lysine acetate transaminase (GOT). The results are expressed as mean ± SD (n = 4 to 6). *** indicates that ρ < 〇. 〇〇 ι 〇 Figure 7B shows the coconut The effect of aqueous extracts on liver injury in a mouse model of hepatic inflammation induced by D_GalN/LPS. Determine the activity of arginine pyruvate transaminase (GPT). The results are expressed as mean ± S D (n = 4 to 6). ** means p<0.01. Figure 7C shows a coconut water extract induced at D_GalN/Lps

The effect of a liver inflamed mouse model on TNF_a performance. The amount of mRNA of ΤΝρ·α is determined by real-time PCR. The results are expressed as mean cuts (4) to 6). *** means p<〇 〇〇1. The graph of Fig. 8 does not show the effect of the phase P water extract on the survival curve of mice after co-injection of LPS/D-GalN. Each group consisted of 6 to 8 mice consisting of a mouse line & intraperitoneal injection of D_Ga called mg/kg body weight) and Lps (Wg/kg body weight). Coconut water extract (OU'Smg/kg body weight) was orally administered prior to D-GalN and milk injection. #图图图9A does not show the effect of coconut water extract on fresh hind limb thickness in a proteoglycan-induced mouse model of Guanlangyan. The control group was given eggs 34 111906 201141500 White polysaccharide to induce arthritis. The normal group was given phosphate buffered saline (PBS) instead of proteoglycan. The treatment group was administered proteoglycan and fed a 0.2, 1 and 5 mg/kg/day dose of coconut water extract. Figure 9B is a graphical representation showing the effect of coconut water extract on the rheumatoid arthritis index in a proteoglycan-induced arthritis mouse model. Coconut water extract was administered at doses of 0.2, 1 and 5 mg/kg/day. [Main component symbol description] None. 35 111906

Claims (1)

201141500 VII. Scope of Application: 1. A method for treating immune diseases and/or abnormalities associated with tumor necrosis factor alpha (TNF-α) on a subject in need thereof, comprising administering a therapeutically effective treatment to a subject in need thereof An amount of coconut water extract or coconut shell extract to induce transforming growth factor/9 (TGF-β) or interleukin 10 (IL-10) as an endogenous immunosuppressive factor. 2. The method according to claim 1, wherein the coconut water extract or the coconut shell extract is prepared by the following method, comprising: (a) passing coconut water or coconut shell water through a resin adsorption type a chromatography column, wherein the coconut shell water is obtained by adding water to the coconut shell; (b) washing the column with water; and (c) being capable of desorbing the coconut water extract from the column or The column is eluted by any solution or combination of solvents of the coconut shell extract. 3. The method of claim 2, wherein the desorption solution or solvent is an organic solvent, water, or any combination thereof. 4. The method of claim 3, wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, acetonitrile, acetone, isopropanol, and any combination thereof. 5. The method of claim 4, wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, and any combination thereof. 6. The method of claim 1, wherein the immune disease and/or abnormality is at least one selected from the group consisting of: inflammation of the skin, inflammation of the liver, rheumatoid arthritis, bonyness Arthritis, knot 1 111906 201141500 Segmental polyarteritis, acute gouty arthritis, asthma, wet repair (ectopic dermatitis), allergic rhinitis, eye allergy, cryoglobulinemia, • primary platelets Reduced purpura, systemic vasculitis, autoimmune hemolytic anemia, Raynaud's phenomenon, systemic lupus erythematosus, scleroderma (systemic sclerosis), insulin-dependent diabetes mellitus, inflammatory bowel disease, Sepsis, septic shock, psoriasis and Behcet's disease. 7. The method of claim 1, wherein the immune disease and/or abnormality is at least one selected from the group consisting of skin inflammation, liver inflammation, and rheumatoid arthritis. 8. The method of claim 1, wherein the subject has skin irritation. ~ 9. The method of claim 2, wherein the subject has liver inflammation. The method of claim 2, wherein the subject has rheumatoid arthritis. ^ The method of claim 1, wherein the therapeutically effective amount of coconut water extract or coconut shell extract has transforming growth factor yS (TGF-泠) or interleukin 10 (11) The induction effect of ^1〇) is used as an endogenous immunosuppressive factor for treating immune diseases and/or abnormalities. 12. The method of claim 2, wherein the coconut water extract or the coconut shell extract comprises at least - a seed, a double enzyme, an oligosaccharide. 13. As stated in the patent application, the towel, the single listener 111906 2 201141500, at least one glucose and mannose. 14. A pharmaceutical composition for the treatment of an immune disease and/or abnormality associated with tumor necrosis factor a (TNF-o:), comprising a therapeutically effective amount of a coconut water extract or a coconut shell extract. 15. The pharmaceutical composition as described in claim 14 is in the form of a lozenge or capsule. 3 111906